While hypoxia-inducible factor (HIF) is a major actor in the cell survival response to hypoxia, HIF also is associated with cell death. Several studies implicate the HIF-induced putative BH3-only proapoptotic genes bnip3 and bnip3l in hypoxia-mediated cell death. We, like others, do not support this assertion. Here, we clearly demonstrate that the hypoxic microenvironment contributes to survival rather than cell death by inducing autophagy. The ablation of Beclin1, a major actor of autophagy, enhances cell death under hypoxic conditions. In addition, the ablation of BNIP3 and/or BNIP3L triggers cell death, and BNIP3 and BNIP3L are crucial for hypoxia-induced autophagy. First, while the small interfering RNA-mediated ablation of either BNIP3 or BNIP3L has little effect on autophagy, the combined silencing of these two HIF targets suppresses hypoxia-mediated autophagy. Second, the ectopic expression of both BNIP3 and BNIP3L in normoxia activates autophagy. Third, 20-mer BH3 peptides of BNIP3 or BNIP3L are sufficient in initiating autophagy in normoxia. Herein, we propose a model in which the atypical BH3 domains of hypoxia-induced BNIP3/BNIP3L have been designed to induce autophagy by disrupting the Bcl-2-Beclin1 complex without inducing cell death. Hypoxia-induced autophagy via BNIP3 and BNIP3L is clearly a survival mechanism that promotes tumor progression.

Hypoxia‐inducible factor (HIF), a transcriptional complex conserved from Caenorhabditis elegans to vertebrates, plays a pivotal role in cellular adaptation to low oxygen availability. In normoxia, the HIF‐α subunits are targeted for destruction by prolyl hydroxylation, a specific modification that provides recognition for the E3 ubiquitin ligase complex containing the von Hippel–Lindau tumour suppressor protein (pVHL). Three HIF prolyl‐hydroxylases (PHD1, 2 and 3) were identified recently in mammals and shown to hydroxylate HIF‐α subunits. Here we show that specific ‘silencing’ of PHD2 with short interfering RNAs is sufficient to stabilize and activate HIF‐1α in normoxia in all the human cells investigated. ‘Silencing’ of PHD1 and PHD3 has no effect on the stability of HIF‐1α either in normoxia or upon re‐oxygenation of cells briefly exposed to hypoxia. We therefore conclude that, in vivo, PHDs have distinct assigned functions, PHD2 being the critical oxygen sensor setting the low steady‐state levels of HIF‐1α in normoxia. Interestingly, PHD2 is upregulated by hypoxia, providing an HIF‐1‐dependent auto‐regulatory mechanism driven by the oxygen tension.

Introduction Intracellular pH regulation controls energy balance and cell proliferation: chemical and biological proof of principle Oncogene activation and transformation cause acidosis Hypoxia promotes acidosis by shifting from oxidative phosphorylation to glycolytic metabolism Hypoxia enhances the expression and activity of pHi‐regulating systems to promote cell survival and invasion Strategies taking advantage of changes in the oxygen level, energy balance and pH homeostasis to target primary tumours and metastases Conclusion Maintenance of cellular pH homeostasis is fundamental to life. A number of key intracellular pH (pHi) regulating systems including the Na⁺/H⁺ exchangers, the proton pump, the monocarboxylate transporters, the HCO₃⁻ transporters and exchangers and the membrane‐associated and cytosolic carbonic anhydrases cooperate in maintaining a pHi that is permissive for cell survival. A common feature of tumours is acidosis caused by hypoxia (low oxygen tension). In addition to oncogene activation and transformation, hypoxia is responsible for inducing acidosis through a shift in cellular metabolism that generates a high acid load in the tumour microenvironment. However, hypoxia and oncogene activation also allow cells to adapt to the potentially toxic effects of an excess in acidosis. Hypoxia does so by inducing the activity of a transcription factor the hypoxia‐inducible factor (HIF), and particularly HIF‐1, that in turn enhances the expression of a number of pHi‐regulating systems that cope with acidosis. In this review, we will focus on the characterization and function of some of the hypoxia‐inducible pH‐regulating systems and their induction by hypoxic stress. It is essential to understand the fundamentals of pH regulation to meet the challenge consisting in targeting tumour metabolism and acidosis as an anti‐tumour approach. We will summarize strategies that take advantage of intracellular and extracellular pH regulation to target the primary tumour and metastatic growth, and to turn around resistance to chemotherapy and radiotherapy.

Malignant tumors exhibit increased dependence on glycolysis, resulting in abundant export of lactic acid, a hypothesized key step in tumorigenesis. Lactic acid is mainly transported by two H⁺/lactate symporters, MCT1/MCT4, that require the ancillary protein CD147/Basigin for their functionality. First, we showed that blocking MCT1/2 in Ras-transformed fibroblasts with AR-C155858 suppressed lactate export, glycolysis, and tumor growth, whereas ectopic expression of MCT4 in these cells conferred resistance to MCT1/2 inhibition and reestablished tumorigenicty. A mutant-derivative, deficient in respiration (res⁻) and exclusively relying on glycolysis for energy, displayed low tumorigenicity. These res⁻cells could develop resistance to MCT1/2 inhibition and became highly tumorigenic by reactivating their endogenousmct4gene, highlighting that MCT4, the hypoxia-inducible and tumor-associated lactate/H⁺symporter, drives tumorigenicity. Second, in the human colon adenocarcinoma cell line (LS174T), we showed that combined silencing of MCT1/MCT4 via inducible shRNA, or silencing of CD147/Basigin alone, significantly reduced glycolytic flux and tumor growth. However, both silencing approaches, which reduced tumor growth, displayed a low level of CD147/Basigin, a multifunctional protumoral protein. To gain insight into CD147/Basigin function, we designed experiments, via zinc finger nuclease-mediatedmct4andbasiginknockouts, to uncouple MCTs from Basigin expression. Inhibition of MCT1 in MCT4-null, Basigin^highcells suppressed tumor growth. Conversely, in Basigin-null cells, in which MCT activity had been maintained, tumorigenicity was not affected. Collectively, these findings highlight that the major protumoral action of CD147/Basigin is to control the energetics of glycolytic tumors via MCT1/MCT4 activity and that blocking lactic acid export provides an efficient anticancer strategy.